Editing can help build stronger muscles

Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function.

Abstract

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disease caused by point mutations, deletions, or duplications in the DMD gene that cause genetic frame-shift or loss of protein expression (1). Efforts under development to reverse the pathological consequences of DYSTROPHIN deficiency in DMD aim to restore its biological function through viral-mediated delivery of genes encoding shortened forms of the protein, up-regulation of compensatory proteins, or interference with the splicing machinery to “skip” mutation-carrying or mutation-adjacent exons in the mRNA and produce a truncated, but still functional, protein [reviewed in (2)].

The potential efficacy of exon-skipping strategies is supported by the relatively mild disease course of Becker muscular dystrophy (BMD) patients with in-frame deletions in DMD (3, 4), and by the capacity of antisense oligonucleotides (AONs), which mask splice donor or acceptor sequences surrounding mutated exons in DMD mRNA, to restore biologically active DYSTROPHIN protein in mice (5, 6) and humans (7, 8). Yet limitations remain for the use of AONs, including variable efficiencies of tissue uptake, depending on antisense oligonucleotide (AON) chemistry, a requirement for repeated AON injection to maintain effective skipping, and the potential for AON-associated toxicities [(9, 10) and supplementary text].

Here, we sought to address these limitations by developing a one-time, multisystemic approach based on the genome-editing capabilities of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system. This system, originally coopted from Streptococcus pyogenes (Sp), couples a DNA double-strand endonuclease with short “guide” RNAs (gRNAs) that provide target specificity to any site in the genome that also contains an adjacent “NGG” protospacer-adjacent motif (PAM) (11–14), which enables targeted gene disruption, replacement, and modification.

To apply CRISPR/Cas9 for exon deletion in DMD, we first established a reporter system for CRISPR activity by “repurposing” the existing Ai9 mouse reporter allele, which encodes the fluorescent tdTomato protein downstream of a ubiquitous CAGGS promoter and “floxed” STOP cassette (15, 16) (fig. S1A). Exposure to SpCas9, together with paired gRNAs targeting near the Ai9 loxP sites (hereafter, Ai9 gRNAs), resulted in excision of intervening DNA and expression of tdTomato (fig. S1, A, B, and E). We next designed and tested paired gRNAs (hereafter, Dmd23 gRNAs) (fig. S1C) that were directed 5′ and 3′ of mouse Dmd exon 23, which in mdx mice carries a nonsense mutation that destabilizes Dmd mRNA and disrupts DYSTROPHIN expression (17). Finally, we coupled the paired Dmd23 and Ai9 gRNAs using a two-plasmid system that links expression of the CRISPR activity reporter (tdTomato) to genome editing events at the Dmd locus (fig. S1D). In vitro transfection of primary satellite cells from mdx mice carrying the Ai9 allele (hereafter, mdx;Ai9 mice) with SpCas9 + Ai9-Dmd23 coupled gRNAs induced gene editing at both the Ai9 locus, demonstrated by tdTomato expression (fig. S1E), and Dmd locus, detected by genomic polymerase chain reaction (PCR) (Fig. 1A) and confirmed by amplicon sequencing (fig. S1F). Dmd editing was not detected in mdx;Ai9 cells receiving Ai9 gRNAs alone (Fig. 1A), although tdTomato expression was equivalently induced (fig. S1E).

Dystrophic pathology and other muscle injuries activate muscle stem cells (also known as satellite cells), which leads to regenerative responses that add new nuclei to damaged fibers [(2) and supplementary text]. To evaluate AAV-CRISPR gene editing in satellite cells in vivo, we crossed mdx;Ai9 mice with Pax7-ZsGreen animals, in which satellite cells are specifically marked by green fluorescence (22), and we injected these animals intramuscularly or systemically with AAV9 encoding Cre (hereafter, AAV-Cre) or Ai9-CRISPR components. Muscles were harvested 2 weeks later (Fig. 4A) and analyzed by FACS. TdTomato expression was apparent in Pax7-ZsGreen+ satellite cells after local or systemic delivery of AAV-Cre or AAV-Ai9 CRISPR (Fig. 4B and fig. S10, A to C), although excision rates were lower for AAV-Ai9 CRISPR than for AAV-Cre. In vitro differentiation of ZsGreen+ satellite cells from mice receiving intramuscular or systemic AAV-Cre or AAV-Ai9 CRISPR produced tdTomato+ myotubes, demonstrating preservation of myogenic potential in AAV-transduced and gene-edited satellite cells (Fig. 4C and fig. S10D). TdTomato+ gene–edited satellite cells also engrafted recipient mdx muscle and contributed to in vivo muscle regeneration after transplantation (fig. S10E).

We next analyzed Dmd editing in Pax7-ZsGreen+ satellite cells after intramuscular or systemic delivery of AAV-Dmd CRISPR or AAV-Ai9 CRISPR. Satellite cells were isolated by FACS, expanded, and differentiated in vitro (Fig. 4A). RT-PCR revealed a truncated Dmd transcript of the expected size and sequence for gene-edited Dmd in satellite cell–derived myotubes from many of the AAV-Dmd CRISPR-injected muscles but none of the AAV-Ai9 CRISPR-injected muscles (Fig. 4D and fig. S10, F, H, and I). Quantification of exon 23 excision revealed variable efficiencies (fig. S10, G and J), which likely reflected targeting of only a subset of endogenous satellite cells that may be variably represented among the isolated and cultured cells. Finally, genomic PCR and amplicon sequencing confirmed targeted excision at the Dmd locus in satellite cell–derived myotubes (fig. S10K), and capillary immunoassay analysis revealed restored DYSTROPHIN expression (fig. S10L). As expected, injection of AAV-Dmd CRISPR did not induce tdTomato expression in satellite cells or myofibers of mdx;Ai9 mice (fig. S11).

In summary, this study provides proof-of-concept evidence supporting the efficacy of in vivo genome editing to correct disruptive mutations in DMD in a relevant dystrophic mouse model. We show that programmable CRISPR complexes can be delivered locally and systemically to terminally differentiated skeletal muscle fibers and cardiomyocytes, as well as muscle satellite cells, in neonatal and adult mice, where they mediate targeted gene modification, restore DYSTROPHIN expression, and partially recover functional deficiencies of dystrophic muscle. As prior studies in mice and humans indicate that DYSTROPHIN levels as low as 3 to 15% of wild type are sufficient to ameliorate pathologic symptoms in the heart and skeletal muscle (23–26) and that levels as low as 30% can suppress the dystrophic phenotype altogether (27), the restoration of DYSTROPHIN achieved here by one-time administration of AAV-Dmd CRISPR clearly encourages further evaluation and optimization of this system as a new candidate modality for the treatment of DMD (see supplementary text).

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Acknowledgments: We thank the Harvard Department of Stem Cell and Regenerative Biology–Harvard Stem Cell Institute Flow Cytometry Core, the Schepens Eye Research Institute–Massachusetts Eye and Ear Institute Gene Transfer Vector Core, the Parker lab at Harvard and J. Goldstein for technical assistance. Work was funded in part by grants from Howard Hughes Medical Institute and NIH (1DP2OD004345, 5U01HL100402, and 5PN2EY018244) to A.J.W. M.T. is an Albert J. Ryan fellow. F.A.R. is a Junior Fellow at the Harvard Society of Fellows. W.X.Y. was supported by T2GM007753 from the National Institute of General Medical Sciences (NIGMS), NIH. F.Z. is a New York Stem Cell Foundation Robertson Investigator and is supported by National Institute of Mental Health, NIH (5DP1-MH100706) and National Institute of Diabetes and Digestive and Kidney Diseases, NIH (5R01DK097768-03); a Waterman Award from the NSF; the Keck, New York Stem Cell, Damon Runyon, Searle Scholars, Merkin, and Vallee Foundations; and B. Metcalfe. W.L.C. is supported by the National Science Scholarship from the Agency for Science, Technology, and Research (A*STAR), Singapore. G.M.C. is supported for this work by National Human Genome Research Institute (NIGMS), NIH, Centers of Excellence in Genomic Science, P50 HG005550. Content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH. M.T., A.J.W., W.L.C., and G.M.C. are inventors on a patent application (PCT/US15/63181) filed by Harvard University related to in vivo genetic modifications and gene editing in muscle. G.M.C. is an inventor on issued patents (US9023649 and US9074199) filed by Harvard University related to CRISPR. L.H.V. is an inventor on a patent application (US2007036760) filed by the University of Pennsylvania related to AAV capsid sequences. F.Z., L.C., F.A.R., and W.Y. are inventors on patents and patent applications (8,865,406; 8,906,616; and accepted EP 2898075, from international patent application WO 2014/093635) filed by the Broad Institute related to SaCas9-optimized components and systems. A.J.W. is an advisor for Fate Therapeutics. G.M.C and F.Z. are founders and scientific advisors of Editas Medicine, and F.Z. is a scientific advisor for Horizon Discovery. G.M.C. has equity in Caribou/Intellia, Egenesis, and Editas (for full disclosure list, see: http://arep.med.harvard.edu/gmc/tech.html). L.H.V. is cofounder, shareholder, member of the scientific advisory board, and consultant for GenSight Biologics, a consultant to Novartis and Eleven Bio, and has received honoraria and consulting fees from Regeneron Pharmaceuticals and Cowen, Jefferies, and Sectoral. AAV9 vector sequences are available through a material transfer agreement (MTA) from the University of Pennsylvania. SaCas9 plasmids are openly available through a Uniform Biological MTA from Addgene.